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Annals. Food Science and Technology

2014

Available on-line at www.afst.valahia.ro Volume 15, Issue 1, 2014 203

DEVELOPMENT OF GAMMA-IRRADIATED LOW MICROBIAL VEGETABLE

SALADS FOR IMMUNOCOMPROMISED PATIENTS

Shubhashis Sarker*1, Mohammad Shakhawat Hussain2, Afifa Khatun

2, Mohammad Afzal Hossain2,

Mohammad Khorshed Alam3 , Mohammad Sabir Hossain

1

*1Department of Biochemistry & Molecular Biology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh 2Food Technology Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Ganak

Bari, Savar, Dhaka-1346, Bangladesh 3Agrochemical & Environmental Research Unit, Institute of Food and Radiation Biology, Atomic Energy Research

Establishment, Ganak Bari, Savar, Dhaka-1346, Bangladesh

*Email: [email protected]

Abstract

Immunocompromised peoples cannot eat raw, uncooked or undercooked foods because of the associated high risk of

infection. One way to overcome this situation is the use of ionizing radiation applied to food. This work covers the

research and development of some salads, which due to the normal risk of adverse microbiological contamination, are

not usually served to immunocompromised hospital patients. Cucumber, tomato, carrot, green leaf lettuce and green

capsicum were treated with 1, 2, 2.5 and 3 kGy radiation from a 60Co gamma irradiator. Changes of the “native”

microflora, and some specific nutritional and physical-chemical properties of irradiated salad vegetables were

analysed. It was observed that generally 1 kGy irradiated samples had less nutritional loss and better sensory score

than the samples irradiated with higher doses. But the initial microflora of the samples were so high that minimum

doses required to meet the sanitary microbiological levels suggested for foods intended for immunocompromised people

and other potential target groups were 2, 2.5, 2.5 and 2 kGy for cucumber, tomato, carrot and green capsicum

respectively. In case of green leaf lettuce the criteria were not met even at above radiation doses. The initial microflora

of the samples will have to be reduced before irradiation to meet microbiological sanitary criteria at low dose

treatment before safely recommend irradiated salads for hospitalized immunocompromised peoples and other target

groups.

Keywords: Microbiological sanitary criteria, irradiation, sensory, biochemical, salads

Submitted: 06.12.2013 Reviewed: 07.02.2014 Accepted: 25.03.2014

1.INTRODUCTION

Fruits and vegetables are important items of a

healthy diet. So, there is an international trend

to increase their consumption (FAO/WHO,

2004). There is an increasing trend in many

countries to centrally prepare and process fresh

fruits and vegetables for distribution and

marketing. Since vegetables are often grown,

processed or packed in areas that may be

exposed to microbial pathogen contamination,

there is an increasing concern that fresh, pre-

cut vegetables may harbor microbial pathogens

(IAEA, 2006). Fresh vegetables at harvest have

a natural epiphytic microflora much of which is

non-pathogenic. During any of the steps-

growth, harvest, processing, packaging,

transportation, handling, retail etc. further

microbial contamination can occur from a

variety of sources, e.g. environmental, animal

or human. There is a risk that this may include

pathogens (FAO/WHO, 2008).

Prepared salads are generally considered safe

to eat by consumers (FSA, 2007). Salad

preparation often involves handling of pre-

cooked or ready-to-eat ingredients with little or

no further cooking steps to reduce the

microbial risk. Two main pathways to

contaminate the final product are- improper

handling of ingredients and contamination after

processing. Fresh vegetables have been

implicated as vehicles for the transmission of

microbial food-borne disease worldwide

(Beuchat, 2006). Problems linked with

pathogens in fresh produce, including the

associated public health and trade implications,

2014 Valahia University Press

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mailto:[email protected]


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have been reported in a number of countries

worldwide (FAO/WHO, 2008). In recent years

the importance of prepared salads as potential

vehicles of infection has been highlighted by

several large outbreaks both nationally and

across international boundaries (Little and

Gillespie, 2008).

Although everyone is susceptible to food-borne

diseases, certain segments of the population are

particularly at risk of contracting a food-borne

illness, namely the immunocompromised,

infants, young children, the elderly, pregnant

women, travelers (WHO, 2000), astronauts,

post-operative patients (IAEA, 2010) etc.

Immunocompromised hospital patients are

estimated to be 20% of the total world

population. People with immune-compromised

systems cannot eat several types of food

because of the associated high risk of infection.

Among these foods are fruits, raw vegetables,

raw eggs and food made with them, raw fish,

unpackaged and undercooked meats,

unpasteurized creams and cheeses, ice-cream,

uncooked nuts, and dried fruits (IAEA, 2010).

The immunocompromised are not only more

susceptible to infections, but suffer more

serious sequelae as a result of infection.

Infections of healthy adults with food-borne

pathogens usually result in self-limiting

gastroentritis that does not require antibiotic

therapy. However, the immunocompromised

persons are at increased risk of complications

(septicemia, arthritis, meningitis, pneumonia)

and death, even if the infecting dose is low

(Trevejo et al., 2005). So, ensuring food safety

is especially important for people who have

impaired immune systems (IAEA, 2010).

Sanitary microbiological levels given by the 1st

Research Coordination Meeting (CRP

15052/RO) of International Atomic Energy

Agency (IAEA) suggested for foods intended

for immunocompromised people and other

potential target groups are shown in Table 1.

These criteria have been derived from Brazilian

guidelines, the International Commission on

Microbiological Specifications for Foods

(IAEA, 2010), information in a scientific paper

by Pizzo et al. (1982), European Regulations

on food hygiene and criteria recommended by

Ju-Woon Lee that were certificated for use in

space flight conditions by the Russian Institute

for Biomedical Problems (IAEA, 2010).

Food irradiation is the treatment of food by a

certain type of energy (ICGFI, 1999). Food

irradiation could be beneficial to society in

general and in particular to

immunocompromised patients who require

high sanitary standards and whose diets are

currently restricted to heat treated foods. The

application of irradiation in combination with

other preservative technologies can contribute

to addressing the pressing need for low

microbial diets in a hospital environment for

immunocompromised patients and other target

groups (IAEA, 2010).

Table 1. Sanitary microbiological levels suggested for foods intended for immunocompromised

people and other potential target groups

Reference: IAEA, 2010

Criterion Microbiological quality

colony-forming unit (cfu) per gram unless specified

Aerobic Plate Counts < 500

Listeria spp not detected in 25 g

Salmonella spp not detected in 25 g

Yeast and Mould < 10

Total Coliform < 10

Staphylococcus aureus < 10

Aerobic spore count < 10

Anaerobic spore count < 10


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Recent research undertaken under a CRP

(2002-2006) (IAEA, 2009) on the use of

irradiation to ensure the safety and quality of

prepared meals established that ionizing

radiation, in combination with good

manufacturing practices and refrigeration,

greatly reduces the risk of food-borne diseases

in a wide variety of foods, and results in both

nutritional and psychological benefits for

immunocompromised patients (IAEA, 2010).

The main objectives of the study were:

microbiological quality assessment of fresh-cut

cucumbers, tomatoes, carrots, green leaf lettuce

and green capsicum; use of different irradiation

doses for shelf-life extension of the vegetable

salads and for inactivation of pathogenic

microbial contaminants; evaluation of some

biochemical and nutritional changes in the

irradiated products; evaluation the sensorial

and physical changes in the irradiated products

during refrigerated storage; and identify the

optimum irradiation dose for each of the

product that reduce microbial load and

inactivate pathogens with minimum changes in

sensory and physical quality attributes.

2. MATERIALS AND METHODS

Samples collection

The study was conducted on five commonly

consumed salad vegetables in Bangladesh:

Cucumber (Cucumis sativus L.), tomato

(Solanum lycopersicum L.), carrot (Daucus

carota L.), green leaf lettuce (Lactuca sativa

L.), green capsicum (Capsicum annuum L.).

The samples were purchased from one Kitchen

market of Dhaka city during winter and spring,

2011 (January-May).

Samples preparation

The samples were washed into running tap

water as we wash in home. Then cucumbers

and carrots were first peeled with a sterile

peeler then uniformly sliced with a sterile knife

on a clean sterile chopping board. Sterilization

was done by autoclaving. Tomatoes were only

sliced. Lettuce and capsicum were chopped.

Stems of all samples were removed. The

samples were packed into sterilized (with 15

kGy radiation dose) food grade transparent

low-density polythene (LDPE, 200 gauge) and

then sealed with a sealer (Impulse Sealer, TEW

Electronic Heating Equipment CO. Ltd.,

Taiwan). Packet size was different according to

the amount of sample packed. For five different

irradiation doses (0, 1, 2, 2.5, 3 kGy), there

were five packets for microbiological analysis

containing 30 g of sample each, five packets

for biochemical analysis containing 50 g of

sample each, five packets for sensory quality

analysis each of which containing five small

packets with five slices of sample. 0 means

non-irradiated sample. All the procedures were

done inside laminar hood.

Irradiation of samples

Doses were applied to the samples at room

temperature from the Co-60 gamma irradiator

source (Located at Atomic Energy Research

Establishment, Institute of Food and Radiation

Biology, Dhaka, Bangladesh) by calibrating

with dose and time basis on central distance

from source to sample where these were

placed.

Microbiological analysis

The microbial contamination in the samples

and the effect of irradiation treatment on the

microorganisms was analysed by counting the

microbial population on the day of irradiation.

The microbiological procedures used to analyse

were decimal dilution technique followed by

pour plating (Gerard et al., 2004). All the

microbiological procedures were done inside a

laminar hood. 5 g of sample (25 g for Listeria

spp.) was homogenized by a autoclaved mortar

and pestle and filtered through a sterile muslin

cloth to a conical flask with 50 ml saline (0.9%

NaCl) water (previously sterilized) to prepare

the stock sample. For the enumeration of total

aerobic spore these suspensions were heated at

80°C for 10 min in a water bath. 1 ml sample

from conical flask was taken in a test tube

containing 9 ml of previously sterilized saline

water. Thus 10-1

dilution was got. This

procedure was repeated where further dilution

was required. With the help of micropipette, 1

ml of the sample from the test tube was poured


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into Petri dishes then sterilized specific media

was poured into Petri dishes and shaken

horizontally to spread out the sample uniformly

over the media. After solidification of the

media the Petri dishes were covered with lids.

Then the Petri dishes were placed in upturned

position in incubator at 37°C (30°C for yeast

and mold) for 24-48 hr.

The analyses were enumeration of total aerobic

flora, total anaerobic bacteria, total aerobic

spore, total yeast and mold, total coliform,

Listeria spp. and Staphylococcus aureus. For

microbiological purposes Nutrient Agar,

Thioglycollate media, Potato Dextrose Agar,

MacConkey Agar, Mannitol Salt Agar were

purchased from Scharlau Chemie S.A. (Spain).

Listeria Selective Agar Base (Oxford

formulation) was purchased from Oxoid LTD

(England).

For anaerobic bacteria Thioglycollate media

was used. After spreading, plates were kept

into an anaerobic jar. The lid of the jar was

closed. After that a vacuum pump was attached

to one port of the jar, and a nitrogen source was

attached to another port of the jar. Then the air

inside the jar was sucked out with vacuum

pump and the jar was filled with nitrogen gas

to maintain anaerobic condition inside the jar.

Then the jar was put inside the incubator.

Biochemical and nutritional analysis

Determination of moisture content

The change of weight was estimated under

certain temperature. The moisture content was

determined by drying the sample at some

elevated temperature and reporting the loss in

weight as moisture (AOAC, 1975).

Determination of ash content

Ash content in the sample was determined

according to Carpenter (1960). About 5-10 g of

the macerated sample was weighted into a pre-

weighted crucible. The crucible with the

content was heated first over a low flame till all

the material was completely charred. Then

charred sample was kept in an Electric Muffle

Furnace (Model no.L9/11/C6, Nabertherm,

Germany) for 4-5 hours at about 600°C for

ashing completely. To ensure the completion of

ashing the crucible was again heated for half an

hour, cooled and then weighed. The weighed

residue was reported as ash.

Determination of ascorbic acid

The estimation of ascorbic acid content was

carried out by the titration result of the sample

extract with 2, 6-Dichlorophenol-Indophenol

dye (BDH Chemicals Ltd., England). The dye

which is blue in alkaline solution and red in

acid solution is reduced by ascorbic acid to

colourless form. The reaction is quantitative

and practically specific for ascorbic acid in

solutions between the pH ranges 1-3.5 (The

association of vitamin chemists, 1966;

Johnson, 1948).

Determination of total carotenoid and

chlorophyll

Total carotenoid and chlorophyll was estimated

by non-maceration method. Total carotenoid

and chlorophyll was extracted in 80% acetone

and the absorption at 663, 645 and 480 nm read

in a spectrophotometer. Using the absorption

coefficients, the amount of total carotenoid,

chlorophyll “a”, chlorophyll “b” and total

chlorophyll was calculated using formula

(Hiscox and Isrealstam, 1979).

The sample was cut into small pieces (or thin

slices). About 1 g of sample was taken and

grinded to a fine pulp in mortar and pestle with

about 10 ml of 80% acetone (MERK,

Germany). The pulp was centrifuged

(Laboratory centrifuge, Model 800, China) at

1790 x g for 5 min. The supernatant was

filtered to a 25 ml volumetric flask. The

sediment in the centrifuge tube was scraped

and ground it again in the same mortar and

pestle with a small amount of 80% acetone.

The mixture was centrifuged as done earlier

and the extract was filtered in the 25 ml

volumetric flask (containing the previous

supernatant). The homogenate was washed out

three to four times with 5 ml of 80% acetone

each time. The final volume was made to 25 ml

with 80% acetone. The extract was kept in

refrigerator for 10 min to lower the

temperature. The absorbance of the extract was


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taken in spectrophotometer (SYSTRONICS,

UV-Vis spectrophotometer, 118, Sr No. 1064,

India) at 663, 645 and 480 nm using 80%

acetone as the blank.

The absorbance of the carotenoids at 480 nm

was determined using the equations provided

by Kirk and Allen (1965). This equation

compensates for interference at this from

chlorophyll. To quantify the carotenoids, Price

and Hendry (1991) and Venkatarayappa et al.

(1984) followed the following equation:

Total carotenoid (mg/g) = A480 + (0.114 × A663)

– (0.638 × A645) × V/1000 × W

Where, A= Absorbance at given wavelength

V= Final volume of 80% acetone in ml

W= Weight of the sample in grams

The amount of chlorophyll “a”, “b” and total

are determined using the following formulas

given by Arnon (1949) based on the work of

Mackinney (1941) who provided the values of

extraction coefficients.

Chlorophyll “a” (mg/g) =

(12.7 × A663) – (2.69 × A645) × V/1000 × W

Chlorophyll “b” (mg/g) =

(22.9 × A645) – (4.68 × A663) × V/1000 × W

Total chlorophyll “a”+ “b” (mg/g) =

(8.02 × A663) + (20.2 × A645) × V/1000 × W

Where, A= Absorbance at given wavelength

V= Final volume of 80% acetone in ml

W= Weight of the sample in grams

Sensory analysis

Method developed by Peryam and Pilgrim

(1975) was used for sensory evaluation.

Following nine points of hedonic scale was

used for sensory evaluation by five judges

(Miyauchi et al., 1964):

9= Like extremely 4=Dislike slightly

8= Like very much 3= Dislike

7= Like 2=Dislike very much

6= Like slightly 1=Dislike extremely

5= Neither like nor dislike

Average sensory score 5 (neither like nor

dislike) is usually acceptable in organoleptic

evaluation. But because we were intended to

supply salads for hospitalized

immunocompromised patients and other

potential target groups, we had to make sure

that they will like the food. So, the

acceptability threshold we considered was

around 7, which means “like” in hedonic scale.

Sensory quality attributes including colour,

flavour, taste and texture of minimally

processed cucumber, tomato, carrot, green leaf

lettuce and green capsicum were evaluated

immediately after irradiation and during

refrigeration (4 ± 1°C) storage. Our intention

was to supply foods as early as possible after

irradiation. So, two days sensory scores were

observed.

Statistical analysis

Results were expressed as mean ± SD

(Standard deviation of mean). One way

ANOVA was performed for data analysis.

ANOVA was followed by Fisher‟s Protected

Least Square Differences (PLSD) for post hoc

comparisons. The statistical program used was

StatView®

5.0 (MindVision Software, Abacus

Concepts, Inc., Berkeley, CA, USA). p<0.05

was considered statistically significant.

3. RESULTS AND DISCUSSIONS

Microbiological analysis

The extent of contamination by

microorganisms in cucumber, tomato, carrot,

green leaf lettuce and green capsicum and the

effect of different doses of gamma radiation

treatment on the contaminated microorganism

level on the day of analysis were determined.

Effect of irradiation on total aerobic plate

count

Initial total aerobic plate counts were around

5.244, 3.505, 6.352, 6.350, 5.185 log cfu/g in

cucumber, tomato, carrot, green leaf lettuce and

green capsicum respectively. Khan et al.

(1992) were collected fresh samples of

cucumber, carrot and lettuce from different

markets in Dhaka metropolitan city,

Bangladesh. Bacterial loads were found to be

7·1x104 to 6·34x10

8 cfu/100 g. From our study


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around 3.2x105 to 2.25x10

8 cfu/100 g of

aerobic bacterial load was observed initially

after washing. Significant differences (p<0.05)

were detected comparing the control with the

results obtained for the irradiated samples

(Table 2). At 1 kGy radiation dose, cucumber,

tomato, carrot, green leaf lettuce and green

capsicum showed approximately 2.943, 0.391,

2.882, 2.511 and 5.185 log cfu/g decrease

respectively. At 2 kGy, cucumber, tomato,

carrot, green leaf lettuce showed approximately

5.244, 0.903, 6.352, 2.8 log cfu/g decrease

respectively. At 2.5 kGy tomato and green leaf

lettuce showed approximately 3.505 and 3.095

log cfu/g decrease respectively.

Microbiological criteria for

immunocompromised people and other

potential target groups (Table 1) were met at

radiation dose 1, 2.5, 2 and 1 kGy for

cucumber, tomato, carrot and green capsicum

respectively. In green leaf lettuce the criteria

were not met at even 2.5 kGy dose.

Effect of irradiation on total anaerobic plate

count

Initial total anaerobic plate counts were around

4.349, 6.114, 4.916, 5.267 log cfu/g in

cucumber, carrot, green leaf lettuce and green

capsicum respectively. No colonies were

detected on tomato. At 1 kGy radiation dose

total reduction of anaerobic bacteria were

observed in every sample (Table 3).

Effect of irradiation on total aerobic spore

count

No spores were detected in any sample except

green leaf lettuce (Table 4). Initial total aerobic

spore count was around 2.845 log cfu/g in

green leaf lettuce. At dose 1 kGy, around 0.845

log cfu/g reduction was observed. At dose 2

kGy, no colonies were detected.

Table 2. Effect of irradiation treatment on total aerobic plate count

Results are expressed as mean ± SD (Standard deviation) [n=3 replicates of each sample for each radiation

dose]. Values in the same row with different superscripts are significantly different at p<0.05 [One-way

ANOVA followed by Fisher‟s PLSD for post hoc comparisons]. cfu/g: Colony forming unit per gram, -: No

colony detected (Detection limit ≥10 cfu/g)

Table 3. Effect of irradiation treatment on total anaerobic plate count





×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 175.5±51.619a 0.2±0.283b - - -

Tomato 3.2±1.697a 1.3±1.697a 0.4±0.283b - -

Carrot 2250±282.843a 2.95±.778b - - -

Green leaf lettuce 2240±212.132a 6.9±1.131b 3.55±0.495b 1.8±0.283b 2.15±0.212b

Green capsicum 153±9.899a - - - -

×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 22.35±1.061a - - - -

Tomato - - - - -

Carrot 1300±28.28a - - - -

Green leaf lettuce 82.5±14.849a - - - -



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Table 4. Effect of irradiation treatment on total aerobic spore count





Table 5. Effect of irradiation treatment on total yeast and mould count





Table 6. Effect of irradiation treatment on total coliform count





×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber - - - - -

Tomato - - - - -

Carrot - - - - -

Green leaf lettuce 0.7±.283a 0.1±0.0b - - -

Green capsicum - - - - -

×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber - - - - -

Tomato - - - - -

Carrot - - - - -

Green leaf lettuce 0.6±0.283a - - - -

Green capsicum - - - - -

×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 19.0±2.970a - - - -

Tomato 0.15±0.071a - - - -

Carrot 1975±176.777a 0.2±0.141b 0.1±0.0b - -

Green leaf lettuce 127.5±7.778a 0.15±0.212b - - -



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Effect of irradiation on total yeast and

mould count

No yeast and mould was detected in any

sample except green leaf lettuce (Table 5).

Initial total yeast and mould count was around

2.778 log cfu/g in green leaf lettuce. At dose 1

kGy, total reduction was observed.

Basbayraktar et al. (2006) observed that the

fungal count of control sample was 5.76 log

cfu/g initially and the fungal count of 1 kGy

irradiated sample was 3.70 log cfu/g. Our study

also showed around 2 log cfu/g decrease of

fungal count at 1 kGy. Bibi et al., (2006)

observed that a dose of 2 kGy in tomato

samples was effective in lowering fungal

colony to safe limits even after 14 days of

storage.

Effect of irradiation on total coliform count

Significant differences (p<0.05) were detected

comparing the control with the results obtained

for the irradiated samples (Table 6). Initial total

coliform counts were approximately 4.279,

2.176, 6.296, 5.106, 5.371 log cfu/g in

cucumber, tomato, carrot, green leaf lettuce and

green capsicum respectively. At 1 kGy

radiation dose, cucumber, tomato and green

capsicum showed total reduction of coliform

whereas carrot and green leaf lettuce showed

around 3.995 and 2.93 log cfu/g decrease

respectively. At 2 kGy, carrot showed

approximately 4.296 log cfu/g reduction. No

coliform was detected in green leaf lettuce at 2

kGy. At 2.5 kGy, carrot showed total reduction

of coliform. Microbiological criteria (Table 1)

were met at radiation dose 1 kGy in cucumber,

tomato and green capsicum. In green leaf

lettuce and carrot, the criteria were met at 2

kGy and 2.5 kGy respectively. Basbayraktar et

al. (2006) observed that the dose of 1.0 kGy

resulted in the 3 log-cycle reduction of E. coli

in different minimally processed fruits and

vegetables. Hammad et al. (2006) predicted

that, the 5 log reduction of E. coli in fresh

produce could be achieved by about 0.55 –

1.55 kGy. We also observed approximately 3

to 5 log-cycle reduction of coliform at 1 kGy.

Effect of irradiation on total Listeria spp.

count

Initial total Listeria spp. counts were around

5.207, 7.122, 6.051, 5.65 log cfu/25 g in

cucumber, carrot, green leaf lettuce and green

capsicum respectively. At 1 kGy radiation

dose, cucumber and green capsicum showed

total reduction of Listeria spp. (Table 7).

Carrot and green leaf lettuce showed

approximately 3.724 and 1.612 log cfu/g

decrease respectively. At 2 kGy, carrot showed

total reduction. At 2 kGy, 2.5 kGy and 3 kGy,

green leaf lettuce showed around 2.176, 2.653

and 2.953 log cfu/g reduction respectively.

Basbayraktar et al. (2006) observed that the

dose of 1.0 kGy resulted in the 3 log-cycle

reduction of L. monocytogenes count. Hammad

et al. (2006) predicted that, 2.6–3.3 kGy should

inactivate 5 log cycles of Listeria

monocytogenes. We also observed around 1-5

log reduction of Listeria sp. at 1-3 kGy.

Microbiological criteria (Table 1) were met at

radiation dose of 1 kGy, in cucumber and green

capsicum. In carrot, at 2 kGy the criteria were

met but in green leaf lettuce the criteria were

not met at our given radiation doses.

Effect of irradiation on Staphylococcus

aureus count

Initial total Staphylococcus aureus counts were

approximately 4.19, 3.161, 2.602, 4.602 and

3.161 log cfu/g in cucumber, tomato, carrot,

green leaf lettuce and green capsicum

respectively. Significant differences were

detected comparing the control with the results

obtained for the irradiated samples (Table 8).

At 1 kGy radiation dose, cucumber showed

total reduction; tomato, carrot, green leaf

lettuce and green capsicum showed around

0.86, 0.602, 1.824, 0.763 log cfu/g reduction

respectively. At 2 kGy, tomato, carrot and

green capsicum showed total reduction. At 2,

2.5 and 3 kGy green leaf lettuce showed 2.204,

2.602 and 2.903 log cfu/g reduction

respectively. Basbayraktar et al. (2006) and

Hammad et al. (2006) predicted that, the 5 log

reduction of Staphylococcus aureus could be

achieved by about 2.1 – 2.7 kGy in different

minimally processed fruits and vegetables.


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Table 7. Effect of irradiation treatment on total Listeria spp. count

Results are expressed as mean ± SD (Standard deviation) [n=3 replicates of each sample for each radiation dose].

Values in the same row with different superscripts are significantly different at p<0.05 [One-way ANOVA followed by

Fisher‟s PLSD for post hoc comparisons]. cfu/25g: Colony forming unit per 25 gram, -: No colony detected (Detection

limit ≥2 cfu/g)

Table 8. Effect of irradiation treatment on Staphylococcus aureus count



Fisher‟s PLSD for post hoc comparisons]. cfu/g: Colony forming unit per gram, -: No colony detected (Detection limit

≥10 cfu/g)

Table 9. Effect of irradiation treatment on moisture content



Fisher‟s PLSD for post hoc comparisons]

Table 10. Effect of irradiation treatment on total ash content




×104 cfu/25g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 16.125±0.53a - - - -

Tomato - - - - -

Carrot 1325±318.198a 0.25±0.0b - - -

Green leaf lettuce 112.5±21.213a 2.75±2.475b 0.75±0.353b 0.25±0.353b 0.125±0.178b

Green capsicum 44.625±19.623a - - - -

×103 cfu/g

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 15.5±2.121a - - - -

Tomato 1.45±0.778a 0.2±0.283b - - -

Carrot 0.4±0.283a 0.1±0.0a - - -

Green leaf lettuce 40±0.0a 0.6±0.141b 0.25±0.071c 0.1±0.141c 0.05±0.071c

Green capsicum 1.45±0.212a 0.25±0.354b - - -

%

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 98.05±0.17a 97.84±0.72a 97.39±0.34a 97.66±0.24a 97.34±0.05a

Tomato 95.89±0.32a 95.58±0.06a 95.64±1.06a 95.14±0.03a 94.88±0.16a

Carrot 92.26±0.16a 91.29±0.29a 91.57±0.62a 90.76±0.11a 92.14±0.55a

Green leaf lettuce 96.2±0.14a 95.67±0.41a 95.5±0.09a 96.32±0.1a 95.73±0.58a

Green capsicum 94.7±0.13a 94.78±0.02a 94.83±0.07a 94.67±0.06a 94.56±0.3a

g/100g of fresh weight of edible food

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 0.310±0.031a 0.272±0.013ab 0.229±0.021b 0.275±0.016ab 0.256±0.012b

Tomato 0.568±0.011a 0.524±0.057a 0.526±0.004a 0.532±0.008a 0.538±0.040a

Carrot 0.475±0.010a 0.460±0.006a 0.459±0.023a 0.475±0.004a 0.480±0.001a

Green leaf lettuce 0.461±0.161a 0.260±0.013b 0.251±0.002b 0.273±0.017ab 0.266±0.023b

Green capsicum 0.203±0.012a 0.268±0.021b 0.268±0.004b 0.283±0.004b 0.292±0.039b


2014


Table 11. Effect of irradiation treatment on ascorbic acid content




Table 12. Effect of irradiation treatment on total carotenoid content




Table 13. Effect of irradiation treatment on chlorophyll “a”, “b” and total chlorophyll content



Fisher‟s PLSD for post hoc comparisons]. Chl: Chlorophyll, -: No chlorophyll detected

mg/100g fresh weight of edible food

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 6.486±0.374ab 5.737±0.098bc 5.196±0.565ce 7.391±0.0ad 4.553±0.643e

Tomato 6.972±0.0a 6.891±0.0a 6.811±0.0a 6.759±0.0a 6.798±0.0a

Carrot 4.643±0.219ab 5.376±0.272b 4.144±0.374ac 4.184±0.296ac 3.250±0.270d

Green leaf lettuce 3.786±0.765a 3.279±0.0a 2.292±0.0b 2.322±0.0b 2.175±0.0b

Green capsicum 4.982±0.641a 3.906±0.0ab 3.839±0.0ab 3.378±0.682b 3.241±0.655b

µg/g fresh weight of edible food

Dose

Samples

0 kGy

1 kGy

2 kGy

2.5 kGy

3 kGy

Cucumber 0.328±0.057a 0.249±0.023ab 0.226±0.097ab 0.143±0.032b 0.181±0.017b

Tomato 2.25±0.071a 2±0.141a 2.3±0.283a 2.45±0.071a 2.4±0.283a

Carrot 6.869±2.49a 6.665±0.615a 5.043±0.603a 6.056±2.65a 6.321±0.538a

Green leaf lettuce 24.5±0.707a 24.5±0.707a 18±4.243b 17±2.828b 18±0.0b

Green capsicum 3.74±0.057ab 3.014±0.339c 3.265±0.205ac 3.082±0.318c 2.967±0.109c

µg/g fresh weight of edible food

Samples Dose

Chl 0 kGy 1 kGy 2 kGy 2.5 kGy 3 kGy

Cucumber

a 4.3±0.46a 3.31±1.06ab 3.52±1.09ab 1.96±0.09b 2.07±0.06b

b 7.76±1.27a 4.39±1.21b 4.44±1.24b 2.91±0.77b 2.55±0.11b

Total 12.06±0.81a 7.70±2.27b 7.96±2.33b 4.86±0.68b 4.69±0.06b

Tomato

a 1.9±0.8a 1.3±0.1ab 0.7±0.1bc 1.1±0ab 0.6±0.1bc

b 1.7±0.5a 1.3±0.1ab 3.2±0.4ac 3.8±1.1c 4.5±1.1c

Total 3.50±1.27ab 2.50±0.14b 3.85±0.21ab 4.85±1.06ac 5.10±0.99ac

Carrot

a - - - - -

b - - - - -

Total - - - - -

Green leaf

lettuce

a 282±57ab 349±9b 217±51ac 212±30ac 229±21ac

b 93±23ab 104±1b 71±11ac 76±6ab 77±8ab

Total 375.5±79.9ab 452.5±10.61b 288±62.23ac 288±35.36ac 306±28.28ac

Green

capsicum

a 42.5±0.71a 37.55±1.48a 31.1±8.63a 35.5±6.08a 39.2±0.42a

b 15.8±1.13a 17.45±1.34ab 20.5±2.12bc 17.25±2.47ab 16.83±0.18ab

Total 58.3±0.42a 55±0.14a 51.6±6.51a 52.75±8.56a 56.03±0.24a


2014


We also observed approximately 3-4 log

reduction of Staphylococcus aureus at 2-3 kGy.

Microbiological criteria (Table 1) were met at

radiation dose 1 kGy in cucumber; at 2 kGy, in

tomato, carrot and green capsicum. In green

leaf lettuce the criteria were not met at our

given radiation doses.

Effect of irradiation on biochemical and

nutritional quality

Similar to other food processing techniques,

irradiation can induce certain alterations that

can modify the chemical composition and

nutritive values of food. Studies have shown

that the macronutrients such as proteins,

carbohydrates and fat are quite stable to the

doses up to 10 kGy. But some vitamins such as

Thiamin (B1) and vitamins A, C and E are

labile to irradiation (Crawford and Ruff, 1996;

WHO, 1994; Wiendl, 1984; Kilcast, 1994;

Giroux and Lacroix, 1998).

Effect of irradiation on moisture content

The moisture content is often an important

aspect of various foodstuffs as excess moisture

can promote microbial growth, which rapidly

deteriorates the quality of food. From our

study, we observed all the samples had

moisture content of more than 90%. So, they

were very favorable for microbial growth. No

statistically significant changes (p<0.05) were

observed in irradiated samples comparing to

the non-irradiated samples (Table 9).

Cucumber had the highest and carrot has the

lowest moisture content.

Effect of irradiation on total ash content

Irradiation‟s effect on permeability and

functionality of cell membranes can result in

electrolyte leakage and loss of tissue integrity.

These effects are limited at dose levels below 1

kGy, but at higher dose levels, electrolyte

leakage may cause a soggy and wilted

appearance. The increase in electrolyte leakage

varies among vegetables. No statistically

significant change (p<0.05) in ash content was

observed in tomato and carrot (Table 10). In

cucumber, significant decrease of ash content

was observed at dose 2 and 3 kGy. In green

leaf lettuce significant decrease of ash content

was observed at dose 1, 2 and 3 kGy. In green

capsicum statistically significant increase of

ash content was observed in irradiated samples.

Around 11.29-17.419% decrease of ash content

was observed in cucumber due to irradiation.

43.601-45.553% decrease of ash content was

observed in green leaf lettuce. And 32.02-

43.842% increase of ash content was observed

in green capsicum. The decrease in ash content

may happen due to electrolyte leakage from the

samples because of irradiation. Fan et al.

(2006) saw that electrolyte leakage increased

linearly with increasing radiation dose for all

vegetables. All vegetables had radiation

thresholds of at least 0.6 kGy. The dose

thresholds for most of the fresh-cut vegetables

were between 1 and 2 kGy. For green leaf

lettuce 1.3 kGy and carrots 0.6 kGy. Green leaf

lettuce at 3 kGy 4.1% and Carrots 9.1%

electrolyte leakage was observed.

Effect of irradiation on ascorbic acid content

Statistically significant (p<0.05) decrease of

ascorbic acid was observed at 2 and 3 kGy in

cucumber (Table 11). No significant change

was observed in tomato. Statistically

significant decrease was observed at 3 kGy in

carrot; at 2, 2.5 and 3 kGy in green leaf lettuce

and at 2.5 and 3 kGy in green capsicum. In

cucumber, ascorbic acid content was decreased

11.548-29.803% due to irradiation except dose

2.5 kGy. At 2.5 kGy, ascorbic acid was

increased 13.953%. At 1 kGy, carrot showed

approximately 15.787% increase then 9.886-

30.002% decrease due to irradiation. 13.391-

42.552% decrease of ascorbic acid was

observed in green leaf lettuce due to

irradiation. In green capsicum, 21.598-

34.946% decrease was observed due to

irradiation. Fan and Sokorai (2002) observed

that at low dose levels (≤1 kGy), irradiation can

reduce ascorbic acid (vitamin C) in some

vegetables, but the decrease is generally

insignificant, given the natural variation

observed in fresh produce, and does not exceed

the decrease seen during storage. Fan and


2014


Sokorai (2005) showed that irradiation

converts ascorbic acid to dehydroascorbic acid,

both of which exhibit biological activity and

are readily interconvertible.

Effect of irradiation on total carotenoid

content

Carotenoids are a class of vegetal pigments,

and some of them can be converted to vitamin

A in human body. The most important

precursor of vitamin A is β-carotene, a

carotenoid with the highest pro-vitamin A

activity (Oliveira and Marchini, 1998). .

Bandekar et al. (2006) observed that there was

no significant difference (p<0.05) in total

carotenoids in the radiation processed (1-2

kGy) and control carrot and cucumber.

Variation in the content carotenoids during

storage also was not statistically significant

from the control samples. We observed

statistically significant (p<0.05) decrease of

total carotenoid at 2.5 and 3 kGy in cucumber

(Table 12). No significant change was observed

in tomato and carrot. Green leaf lettuce was

showed significant decrease of total carotenoid

at 2, 2.5 and 3 kGy. Green capsicum showed

statistically significant decrease of total

carotenoid at 1, 2.5 and 3 kGy. In cucumber,

total carotenoid content was decreased 24.085-

56.402% due to irradiation. In green leaf

lettuce, no change in total carotenoid content

was observed at dose 1 kGy. Around 26.531-

30.612% decrease in total carotenoid content of

green leaf lettuce was observed due to radiation

dose 2-3 kGy. In green capsicum,

approximately 12.701-20.668% decrease of

total carotenoid content was observed due to

irradiation.

Effect of irradiation on chlorophyll content

Salunkhe (1956); Wishnetsky et al. (1959)

showed chlorophylls are sensitive to

irradiation. A linear decrease in the chlorophyll

content of green beans and broccoli was

directly related to the absorbed dose of 4.9 to

92.9 kGy (Wishnetsky et al., 1959). Our study

showed no significant total chlorophyll

reduction except cucumber at 1-3kGy.

Irradiation temperatures, type of vegetables,

and headspace atmosphere have an influence

on degradation of carotenoids and chlorophylls

(Franceschini et al., 1959). No chlorophyll was

detected in carrot (Table 13). Irradiated tomato,

green leaf lettuce and green capsicum didn‟t

show statistically significant changes of total

chlorophyll content compared to control.

Effect of irradiation on sensory quality

Historically, the high radiation doses used in

attempts to produce a sterile or shelf-stable

fruit or vegetable commodity have resulted in

unpalatable products. Irradiation may induce

the loss of firmness (softening) in some fruits

(Gunes et al., 2000; Palekar et al., 2004). On

low dose levels (1 kGy or less), most fresh-cut

vegetables show little change in appearance,

flavor, color, and texture, although some

products can lose firmness. From the sensory

quality analysis we observed the change in

sensory quality mainly loss of firmness at

higher irradiation dose than 1 kGy. Mohácsi-

Farkas et al. (2006) observed 1 kGy was

acceptable radiation dose for the treatment of

pre-cut tomato, having no significant effect on

sensory properties, firmness and antioxidant

vitamins.

Basbayraktar et al. (2006) observed that at 1.0

kGy irradiation lack of adverse effects on

sensory attributes. Bibi et al. (2006) observed

that a dose of 2 kGy for carrots, 2.5 kGy for

cucumber was sufficient to keep them

microbiologically and sensorially acceptable

for two weeks at refrigerated temperature.

Tomato due to the soft texture and microbial

spoilage cannot be stored up to two weeks, a

dose of 2.5 kGy for tomato can be

recommended for the refrigerated storage up to

one week. No significant (p<0.05) radiation

induced change in sensory quality of cucumber

on the day of irradiation.

On day 1, all scores were over acceptability

threshold (7). On day 2, 2 kGy and 2.5 kGy

irradiated sample‟s texture score was below

acceptability threshold (around 5). In tomato,

softening of texture observed at dose 3 kGy on

the day of irradiation. 3 kGy irradiated tomato

texture score was below acceptability threshold

on day 1.


2014


Figure 1. Changes of overall acceptance of cucumber (a), tomato (b), carrot (c), green leaf lettuce (d) and green

capsicum (e) during two consecutive days of storage at 4°C. Results are expressed as average of sensory scores (colour,

flavour, taste and texture). Sensory scores according to hedonic scale. 7= Acceptability threshold. With the progress of

storage period, overall acceptance was decreased both in non-irradiated and irradiated samples. Carrot, green leaf

lettuce and green capsicum showed better overall acceptance than cucumber and tomato in two days storage period.


2014


On day 2, taste score of 2 and 2.5 kGy tomato

and texture score of 2-3 kGy tomato were

below acceptability threshold. In carrot and

green capsicum, softening of texture was

observed at dose 2-3 kGy on the day of

irradiation. With the progress of storage day

sensory scores decrease more in irradiated

samples than control sample (see Figure 1). But

all the sensory scores were over acceptability

threshold in two days of storage. No significant

radiation induced change in sensory quality of

green leaf lettuce was observed on the day of

irradiation. On day 2, significant change in

flavour, taste and texture were observed at dose

1 and 2 kGy. But all the sensory scores were

over acceptability threshold in two days of

storage. On day 2, control, 1 and 2 kGy

irradiated green capsicum‟s texture was below

acceptability threshold. On day 2, overall

acceptance of 2.5 and 3 kGy irradiated

cucumber and 2-3 kGy irradiated tomato was

below acceptability threshold.

Patterson and Stewart (2003) were

experimented the effect of ionizing radiation on

the microbiological and nutritional quality of

chilled ready meals normally produced for

consumption in a number of hospitals in the

Belfast area or for use as „mealson- wheels‟ for

elderly people. Results showed that an

irradiation dose of 2 kGy can be used to extend

the shelf-life of ready meals for up to 14 days

and that the irradiated meals must be stored

under good refrigeration conditions (<3°C) in

order to obtain maximum benefit from the

irradiation treatment. Higher irradiation doses

used for any fresh produce were found to be

better for controlling microbial counts than

lower doses. But a CRP of IAEA was

demonstrated that in general, fruits can be

exposed to doses between 1.0-2.0 kGy without

affecting the sensory attributes. It was also

demonstrated that most of the studied

minimally processed vegetables could be

irradiated with doses up to 2 kGy. These doses

were effective in reducing the initial microflora

in 4–5 logs and at the same time extending the

shelf-life of the products without adverse effect

on their sensory characteristics (IAEA, 2006).

From our observation we may come into

decision that freshly-cut cucumber and tomato

irradiated over 1 kGy should not be

recommended after one day of storage at

refrigeration temperature. Freshly-cut carrot,

green leaf lettuce and green capsicum

irradiated up to 3 kGy could be supplied to the

hospitals between two days of storage. But 3

kGy dose could not make green leaf lettuce

microbiologically safe.

5. CONCLUSIONS

Bangladesh has the seventh largest population

in the world (PRB, 2009). The number of

immunocompromised patients is steadily

increasing due to increased incidence of

diseases like cancer, HIV/AIDS, diabetes, and

increase in age expectancy. There is a need to

develop microbiologically safe and

nutritionally wholesome food products for this

segment of the population (IAEA, 2010).

Like many 3rd

world countries hygienic quality

of the fruits and vegetables of Bangladesh are

maintained so poorly. From this study, it was

also observed, initial microflora of the samples

even after washing with water were so high

that, minimum doses required to meet the

microbiological criteria (Table 1) were 2, 2.5,

2.5 and 2 kGy in cucumber, tomato, carrot and

green capsicum respectively. In green leaf

lettuce the criteria were not met at our given

radiation doses. But our study showed

biochemical, nutritional and sensory quality

altered for some vegetables in those doses.

EFSA Panel on Biological Hazards (2011)

recommend that upper dose limits for pathogen

reduction should not be specified, since other

constraints, such as undesirable chemical

changes, will limit the doses applied. Currently

maximum allowable dose for uncut vegetables

in USA is 1 kGy (EPA, 2013; FAO/IAEA,

2012; FDA, 2007), in Russia 0.03 kGy, in UK

1 kGy (FAO/IAEA, 2012), in EU 1 kGy (FSA,

2012). And advisory technological dose limit

given by IAEA (IAEA, 1998) for self-life

extension of uncut fresh fruits and vegetables

was 2.5 kGy but not for microbial food safety.


2014


So, in Bangladesh supplying ready-to-eat low

microbial vegetable salads to

immunocompromised patients will not be

possible if only irradiation technology is used.

Further research should be done with various

disinfecting treatment to reduce initial

microbial load along with modified atmosphere

packaging before irradiation to meet the

microbiological criteria at low dose irradiation

treatment.

6. ACKNOWLEDGEMENTS

We thank Atomic Energy Research

Establishment, Bangladesh for giving

permission to do this work at Institute of Food

and Radiation Biology. This work was

financially supported by International Atomic

Energy Agency (IAEA) Coordinated Research

Project (CRP) on “Use of Irradiation for Shelf

Stable Sterile Foods for Immunocompromised

Patients and Other target Groups” (Contact No.

15052/RO).

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